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Fluorescent intercalator displacement as a tool for RNA‐ligand discovery
Author(s) -
AsareOkai Papa Nii,
Chow Christine S
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.907.1
Subject(s) - nucleic acid , chemistry , rna , fluorescence , electrospray ionization , small molecule , combinatorial chemistry , ligand (biochemistry) , nucleic acid structure , intercalation (chemistry) , mass spectrometry , chromatography , stereochemistry , biochemistry , organic chemistry , quantum mechanics , gene , physics , receptor
Fluorescent intercalator displacement (FID) is a useful tool for identifying new nucleic‐acid‐binding ligands. The success of this method is based on the fact that it can be fashioned into a versatile high‐throughput assay, which can then be used to assess relative binding affinities of compounds to nucleic acids in a simple and efficient manner. FID is becoming an increasingly attractive method because it is a tag‐less approach; neither the nucleic acid nor the small molecule under investigation has to be modified. In this study, a modified FID method for screening RNA‐binding ligands was established, using 3‐methyl‐2‐((1‐(3‐(trimethylammonio)propyl)‐4‐quinolinylidene)methyl)benzothiazolium (TO‐PRO) as the fluorescent intercalator. Electrospray ionization mass spectrometry (ESI‐MS) was employed to investigate the FID mechanism. Our results provide molecular evidence that correlates a reduction in fluorescence intensity in the FID assay with displacement of the TO‐PRO dye molecule from structured RNA. The FID assay was then used to screen a variety of RNA‐binding ligands. With FID, it is possible to differentiate ligands that bind to a variety of RNA constructs from the ribosome and HIV (A‐site, h31, H69, and TAR) with moderate selectivity. This work is supported by the National Institute of Health (NIH)